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Methylglyoxal, a Diabetic Biomarker for Insulin Resistance, Vascular Dysfunction and Neuropathies

Yousif A Shamsaldeen, Louise S Mackenzie, Lisa A Lione and Christopher D Benham

Abstract: Diabetes mellitus is a pandemic metabolic disease characterized by chronically elevated blood glucose concentration due to insulin dysfunction. Diabetics are prone to limbs fungal infections and ulcers due to vascular injury and end-organ damage such as nephropathy, retinopathy and neuropathic pain all of which are associated with methylglyoxal elevation. Methylglyoxal is generated through carbohydrate, lipid and protein metabolism which are all found to be exacerbated in diabetes. Moreover, methylglyoxal is highly reactive with various cellular and interstitial molecules such as proteins and phospholipids to form stable adducts and advanced glycation end products. Methylglyoxal induces insulin resistance, pancreatic β-cells cytotoxicity, and induces endothelial dysfunction that accelerates retinopathy, another diabetes complication. Additionally, methylglyoxal induces hyperalgesia and neuronal inflammation associated with neuropathic pain. Therefore, methylglyoxal might represent a potential therapeutic target in diabetes and associated complications.

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1. Introduction:

Diabetes mellitus (DM) is a pandemic metabolic disease characterized by chronically

elevated blood glucose concentration (hyperglycaemia) due to insulin dysfunction which is

attributed to insufficient -or blunted- insulin secretion and/or declined tissue sensitivity to

insulin1. The incidence of diabetes mellitus is increasing throughout the world and numbers

are expected to reach 592 million by the year 2035, mainly because of the increase in

obesity2. Approximately 50% of diabetics show diabetes complications by the time when

they are diagnosed3. Approximately 5 million people died from diabetes in 2014 globally

which accounts for a death every 7 seconds4. Despite the differences in aetiology, clinical

presentation, and disease prevalence, secondary complications, such as neuropathic pain,

occur in both type 1 and 2 diabetes mellitus5. Diabetes complications lead to a reduced

quality of life posing a huge economic burden to the health system and society5. In 2011 in

the UK, the NHS spent almost £24 billion on diabetes, 30% of which was spent on managing

complications, such as neuropathic pain. By 2035, it is estimated that diabetes will cost the

NHS approximately £40 billion, accounting for 17% of total health resource expenditure6.

Among diabetic patients approximately 10% are diagnosed with type-1-DM (T1DM) which is

mainly attributed to autoimmune activity where expressed plasma islet-cells antibodies

destroy pancreatic β-cells1, 7. Children, under 12 years comprise the majority of T1DM

patients who require life-long insulin treatment for survival. However, there are two types

of monogenic diabetes which are commonly misdiagnosed as T1DM due to early onset of

disease. These are neonatal diabetes (ND) which is diagnosed in the first 6 months of life,

whereas the second type; maturity-onset diabetes of the young (MODY) affects individuals

younger than 25 years, controlled largely through the use of sulphonylureas such as

glibenclamide7.

The large majority of diabetics, are of type-2-DM (T2DM), which is regarded as a complex

disease that embraces genetic factors, lifestyle, age, obesity, pregnancy and gender as risk

factors8. Unlike T1DM, patients of T2DM usually do not require insulin to survive since

insulin secretion is only partially deficient and/or the individual is insulin resistant. Insulin

resistance is mainly attributed to chronically elevated levels of insulin reducing sensitivity

and further increased by abdominal fat and hence obesity1. Reduced insulin secretion may

be due to insulin signalling cascade alteration and/or reduced pancreatic β-cells mass,

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however, the extent of pancreatic β-cells mass reduction is controversial as some studies

stated that 65% loss of pancreatic β-cells is sufficient to induce diabetes9 while other studies

concluded that 10% reduction of pancreatic β-cells mass is also associated with numerous

altered insulin signalling components that initiate diabetes10.

2. Methylglyoxal and diabetes:

In addition to the previously mentioned diabetes complications, diabetics suffer from

frequent: thirst (polydipsia), urination (polyuria) and hunger (polyphagia)1 , these common

complications are recently found to be associated with plasma/tissue methylglyoxal (MGO)

elevation (Table 1).

Chronic hyperglycaemia is the main DM complication where blood glucose concentration

exceeds 7mmol/L (125mg/dl). This results in an increasing proportion of glucose metabolism

(approximately 0.5% of glycolysis), passing down alternate pathways to generate reactive

oxygen species (ROS) such as MGO that is highly reactive with various cellular and interstitial

molecules such as proteins and phospholipids to form stable adducts and advanced

glycation end products (AGE)11 12. Upon forming AGE, MGO is trapped intracellularly and

subsequently increases oxidative stress (OS) that disrupts the cellular membrane integrity

and thereby allows MGO leakage to the serum from where it can be measured for disease

progression and severity11 13 14. Physiological human plasma MGO concentration is

approximately 150nM and is doubled in T2DM patients’ plasma15. Moreover, glycolysis-

derived MGO interacts with cellular proteins and nucleic acids to accelerate AGE production

resulting in pancreatic β-cell cytotoxicity. This exacerbates hyperglycaemia and hence DM

complications11. However, clinical studies have failed to significantly correlate MGO to blood

glucose concentration due to 2 main technical reasons, (i) firstly it is essential to dissociate

MGO from protein without causing any DNA damage and/or oxidation to accurately

measure total MGO, and (ii) the heterogeneity of the samples due to diverse patient

backgrounds14.

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2.1. MGO sources:

Being an AGE precursor, MGO levels have been widely studied14. The 4 main MGO sources

are summarized in the following formula:

MGO total = MGO carbohydrates + MGO lipids + MGO proteins + MGO exogenous

As shown in figure 1, three main integrated metabolic pathways are involved in MGO

formation:

1. Carbohydrates:

Reducing sugars are able to react with proteins’ amino groups to yield Schiff’s base

which is structurally rearranged into Amadori product to be subjected to series of

reactions that generate AGE12. Accordingly, MGO is generated mainly through

phosphorylating glycolysis such as triose-phosphate enzymatic metabolism which

was found to be increased in hyperglycaemia, the pentose phosphate shunt, sorbitol

pathways such as xylitol metabolism and fourthly glucoxidation14 16. Moreover,

triose-phosphate accumulation is involved in diabetic nephropathy revealing the

involvement of carbohydrate generated MGO pathway in diabetes complication and

such a pathway was shown to be inhibited through thiamine17 18 (figure 1).

2. Lipid pathways:

Lipid peroxidation of polyunsaturated fatty acids yields short chain; 3-9 carbon long

fragment molecules of highly reactive aldehyde such as 2-Alkenol, 4-hydroxy-2-

alkenal as well as ketoaldehydes from which glyoxal compounds such as MGO is

generated from non-enzymatic and enzymatic metabolism of acetoacetate or

acetone intermediates, respectively12 14. Acetoacetate is a major ketone body (KB)

elevated in type 2 diabetics’ plasma19. Moreover, lipolysis shares a common

intermediate with carbohydrate metabolism; triose-phosphate through α-glycero-

phosphate dehydrogenase-metabolized glycerol14. Previous studies found that

lipolysis is increased in diabetes and suppression of lipolysis improves insulin

sensitivity and glucose utilization20 21. Moreover, isopropyl alcohol (IP) is significantly

increased (5mg/dl) in ketoacidosis diabetic plasma22. IP is a metabolite of acetone, as

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the reaction is governed by alcohol dehydrogenase which favours the production of

IP when acetone is increased as in diabetes mellitus where glucose utilization is

compromised, and the body adapts the energy supply toward fatty acid oxidation

through NAD+ reduction into NADH. Therefore, when NADH/NAD+ ratio is increased,

NADH is utilized to produce IP from acetone22 23.

3. Protein metabolism:

Numerous in vitro studies demonstrate the vulnerability of tyrosine, serine,

threonine and glycine rich proteins toward oxidation as these residues are

enzymatically converted to acetone and aminoacetone intermediates which are

converted into MGO12 14. Moreover, semi-carbazide sensitive amine oxidase (SSAO)

which converts aminoacetone into MGO and hydrogen peroxide is the only enzyme

found elevated in diabetic plasma14. Protein catabolism is increased by

approximately 50% in streptozotocin treated rats, and this increased rate of protein

catabolism is attributed to insulin resistance and increased glucocorticoids

production in normal rats24.

4. Exogenous MGO:

AGE are ingested through heated and processed fats, proteins (Maillard reaction) as

well as sugars and tobacco25, whereas previous studies revealed that coffee and

whiskies are the main MGO-containing beverages26 27. The average daily

consumption of AGE is

16000kU AGE (kilo unit AGE per gram of serving size) which is even aggravated by

high temperature processing such as oven frying that increases the AGE content of

chicken breast to 900kU/g compared to boiled chicken breast

(100kU AGE/g)28. Chronic consumption of AGE-containing food/beverages causes

mild liver inflammation as well as fat disposition on parenchymal cells which alters

fasting insulin and thereby reduces glucose tolerance according to previous research

conducted on rat hepatocytes 27. In the Maillard reaction, reducing sugars such as

glucose interact with free amino groups found in proteins to form for instance

N-substituted glycosylamine which in the presence of water goes through Amadori

rearrangement to yield Amadori product, 1-amino-1-deoxy-2-ketose29. The

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rearranged Amadori product (RAP) is then degraded through 2,3 enolisation to form

numerous fission products such as acetol, pyruvaldehyde and diacetyl compounds at

pH˃729. These carbonyl compounds are highly reactive with amino acids to form

aldehydes and α-aminoketones29. Previous studies found that Maillard reaction

products are significantly increased in diabetics’ skin collagen as Nε-carboxymethyl

lysine (CML), fructoselysine (FL) and pentosidine which are associated with

accelerated aging30. Additionally, CML is significantly elevated in diabetic plasma

when compared to healthy plasma, and such elevation is exacerbated when purely

prepared AGE beverages were ingested22. This elevation was associated with altered

vascular function through supressing the expression and function of eNOS as well as

stimulating the release of vascular cells adhesion molecules (VCAM-1)25.

2.2. MGO metabolism:

Two glutathione (GSH) dependent pathways contribute to MGO metabolism; glyoxalase

system (GLO), glyoxalase 1 and glyoxalase 2 (GLO1 & GLO2) and aldose reductase, of which

GLO-1 is the major pathway that converts MGO it to non-toxic D-lactate14 31. However, as

11% of glucose is metabolized through the sorbitol pathway, the bi-modal aldose reductase

acts as aldehyde reductase rather than ketone reductase and thereby preferentially

produces acetol which thereafter is converted retrospectively to MGO through CYP2E1 to

start a futile cycle that depletes the intracellular GSH and elevates acetol in diabetic

plasma14.

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Figure 1 Endogenous sources of methylglyoxal (MGO) from glucose, lipid and protein metabolism. (A) Normal

non-diabetic condition shows low MGO production from un-affected glycolysis, lipolysis or proteolysis with

major sources represented in bold arrows. (B) Diabetes is associated with increased MGO production from

hyperglycaemia, accelerated lipolysis and proteolysis represented with thick borders and bold arrows which

are accompanied with compromised glyoxalase activity.

B

A

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2.3. MGO and insulin:

Insulin secretion is a calcium-dependent cascade which starts when pancreatic β-cells

glucose-transporter-1 (GLUT-1) take up glucose due to elevated plasma-glucose. This results

in ATP synthesis and potassium ATP channel (KATP) closure. Once KATP are closed, Calcium

ions (Ca+2) enter through Voltage gated Ca+2 channels initiating insulin exocytosis32.

However, glucose is not the only insulin release stimulator as lipids and proteins are also

insulin secretagogues, in addition to other neurotransmitters and hormones such as

incretins which stimulate insulin secretion independently from Ca+2 as illustrated in figure

27.

Insulin resistance is a complex condition where a normal insulin concentration is not

sufficient to mediate glucose uptake and utilization due to insulin signalling disruption and

hence, more insulin is released to try to maintain glucose homeostasis33 34. MGO has been

shown to bind with insulin through targeting arginine residues located in chain B and at N-

terminus33. Subsequently, MGO-modified insulin chain B is heavier than free insulin by an

additional 126Da resulting in less glucose uptake and utilization. This has been

demonstrated in skeletal muscle L8 cells and 3T3-L1 adipocytes as well as a 50% reduction in

metabolism in H4-II-E hepatocytes33. This study is further supported by the fact that insulin

receptor substrate-1 (IRS-1) phosphorylation and PI3K activity are both suppressed dose

dependently following MGO and reversed with MGO scavenger; N-acetylcysteine34 (Table

1). Moreover, MGO (100μM) induces pancreatic β-cytotoxicity when applied to RINmf5

insulin secreting cells in culture11. Such an effect is comparable to a high glucose (16mM) apoptotic

effect on rat pancreatic β-cells11. These findings suggest that MGO might play a major role in

progressive stages of diabetes where chronic hyperglycaemia yields elevated MGO that reduces

insulin signalling and pancreatic β-cells numbers leading to a further reduction in insulin secretion11

(Figure 2). Moreover, when MGO 60mg/kg/day was infused in Sprague-Dawley rats for 28 days, it

resulted in a significant reduction in plasma insulin and a significant increase in fasting plasma

glucose. Additionally, plasma and pancreatic, muscle and, adipocyte tissues were all characterized by

significant MGO elevation associated with significant decrease in glutathione (GSH) and adipocyte

plasma membrane glucose transporter-4 (GLUT-4) as well as pancreatic GLUT-2 35.

Insulin binds to its corresponding tyrosine-kinase coupled endothelial receptor, insulin receptor (IR)

which is phosphorylated and provides a docking site for insulin receptor substrate-1 (IRS-1) to bind.

As it exerts tyrosine-kinase activity, IR phosphorylates IRS-1 to unveil an interactive sulfhydryl

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(SH2) domain which is responsible for activating phosphatidylinositol 3-kinase /protein

kinase B (PI-3K/PKB) as well as Ras-mitogen activated protein kinase (MAPK) pathways.

These pathways phosphorylate approximately 40 cellular targets including Akt-dependent

endothelial nitric oxide synthase (eNOS) that generates nitric oxide (NO) to yield

endothelium-dependent vasodilation34 36 37 38. Therefore, MGO-induced insulin dysfunction

(reduced secretion and increased resistance) might directly cause vascular dysfunction, a

common complication in diabetes37 (Figure 3).

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Figure 2 Insulin release from pancreatic β-cells through calcium dependent and independent pathways. GLUT-1 takes up glucose which is metabolised through glucokinase (GCK) to glucose-6-phophate which is metabolised in the mitochondria to generate ATP. A rise in [ATP] stimulates the closure of potassium ATP channels and depolarisation that triggers Ca+2 influx. Whereas glucagon like peptide-1 (GLP-1) stimulates insulin secretion via an exchange protein activated by cAMP (EPAC) and protein kinase A (PKA)-dependent mechanism which are activated through cyclic adenosine monophosphate (cAMP). Plus signs reveal stimulation. (A) normal insulin secretion. (B) Methylglyoxal (MGO) is toxic to pancreatic β-cells and forms insulin-adducts which endows insulin with higher molecular weight and less activity.

2.4. MGO and vasculature:

B

A

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Numerous authors correlate highly accumulated MGO to vascular and other end organ

damage such as nephropathy and neuropathy39. Vascular dysfunction is a common

complication in DM which may culminate with renal failure, blindness and limb

amputation40 41.

As a highly reactive aldehyde MGO forms stable adducts when reacting with multiple

macromolecules through preferentially targeted aminoacids as for instance lysine;

Nε-carboxyethyl lysine (CEL), MGO-derived lysine-lysine dimer (MOLD), arginine; 5-

methylimidazolone, tetra-hydropyrimidine and argpyrimidine as well as sulfhydryl group-

containing cysteine which forms stabilized S-lactyl cysteine through keto-enol tautoumerism

from which CEL and MOLD are both elevated in DM12. Accordingly, MGO inhibits eNOS

through inhibiting the phosphorylation of serine 1177, thereby inhibiting NO production and

yielding vascular dysfunction40 (Figure 3). Moreover, rat thoracic aortic smooth muscle cells

(ASMC) treated with MGO (100μM) induces NO production and upon further investigations,

the researchers found that H2O2 generation was also enhanced39. Furthermore, induced NO

reacts with peroxides (O.2) forming the highly reactive oxidant peroxynitrite (ONOO-) so that

NO is bound and thereby physiologically non-functional. In addition, ONOO- itself is

considered as an essential atherosclerotic factor39. Furthermore, immunohistochemical-

based observations in kidneys from diabetic patients show increased argpyrimidine

formation in patient arteries suggesting MGO induces arterial injury as a further DM

complication42. Vascular function restoration in GLO-1-overexpressed STZ treated animals is

accompanied by MGO and AGE reduction and another study showed vascular function

restoration through MGO scavengers such as diacetyl cysteine40 41 (Table 1).

MGO is also a redox-based cell signalling regulator39, which oxidizes GSH to GSSH through

irreversible binding to arginine and thereby alters the cellular redox system which pushed

the cell toward oxidative stress-induced apoptosis33.

MGO blocks insulin-stimulated eNOS phosphorylation at serine-1177 and threonine-497 and

also inhibited tyrosine phosphorylation of IRS-1 and Akt37. GLO-1 inhibition results in

elevation of MGO and showed similar inhibitory effects on eNOS, IRS-1 and Akt

phosphorylation. Moreover, in vivo studies on mice administered i.p MGO 50-75mg/kg/day

for 5 consecutive days/week for 7 weeks showed significant insulin resistance accompanied

with compromised endothelial function that was attributed to IRS-1 inhibition through

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serine-616 phosphorylation and eNOS signalling suppression37 (Figure 3). Additionally,

endothelial dysfunction is accompanied by an increase in oxidative stress markers such as

the vascular monocytes chemoattractant peptide-1 (MCP-1) and RAGE expression in Wistar

and Goto-Kakizaki rats43. Tetrahydropyrimidine (THP), an AGE product of MGO was elevated

in T1DM compared to non-diabetics (115.5U/μl vs 109.8U/μl) and such elevation was

strongly associated with an endothelial dysfunction marker, soluble vascular cell adhesion

molecule-1 (sVCAM-1) and phospholipase-A2 (sPLA2), a low grade inflammatory marker.

This data suggests MGO as a diabetic marker for vascular dysfunction44.

These diabetic vascular complications are commonly associated with retinopathy. MGO-

derived CML, CEL and hydroimidazalone-1 (MG-H1) are increased in diabetic wild type (WT)

rat retina but not in diabetic GLO-1 overexpressing transgenic rats or in non-diabetic rats.

GLO-1 overexpression prevented the generation of new capillaries in acellular tissues in

central and peripheral regions of retina as well as preventing cellular capillary

degeneration45.

In addition to all these vascular complications, the lifespan of erythrocytes (RBC) is reduced

in diabetes. The MGO concentration is doubled in diabetics’ RBC and elevated by fourfold in

diabetics’ plasma compared to non-diabetics’46. MGO accelerates RBC suicidal death,

eryptosis through enhancing phosphatidylserine exposure on the cell surface, a signal that

triggers cell death. Further, MGO was shown to reduce ATP and GSH levels in RBCs which

would accelerates eryptosis46 47. Moreover, ROS increment in haemodialysis patients (HD)

reveals that cell injury is a major factor that corresponds to ROS leakage and plasma ROS

increment, a fact that is supported by recent studies showing ROS such as methylglyoxal

induces RBC injury14 48.

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Figure 3 Methylglyoxal (MGO) induces vascular dysfunction by interfering with insulin signalling. (A) Normal endothelial vasodilation stimulated through insulin binding to its insulin receptor (IR) which is phosphorylated to unveil intracellular binding site for insulin receptor substrate-1 (IRS-1) which is phosphorylated upon binding. Phosphorylated IRS-1 provides a sulfhydryl (SH2) residue which is then bound with phosphatidylinositol 3-kinase (PI3K) that phopsphorylates protein kinase B (PKB) which phosphorylates and activates Akt that activates endothelial nitric oxide synthase to generate nitric oxide (NO). NO diffuses into smooth muscle cells (SMCs) causing vasodilation (VD). (B) MGO reduces insulin secretion through pancreatic β-cells toxicity, binds to insulin forming the less active insulin-MGO adduct and inhibiting eNOS phosphorylation which results in less NO production and compromised endothelial vasodilation.

B

A

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2.5. MGO and neuropathic pain:

Diabetes is one of the leading causes of chronic neuropathic pain 5. Neuropathic pain occurs

in both Type 1 and Type 2 diabetics and given the heterogeneity of mechanisms that drives

neuropathic pain in patients and over time, it is challenging to identify an optimum

treatment strategy5. Among the major diabetic complications is diabetic neuropathy where

nociception is exacerbated due to diabetes13. A recent study found diabetic plasma MGO

was approximately doubled and reached 1µM in diabetic individuals with hyperalgesia

which was associated with increased COX-2 expression and suppressed GLO-1 activity31.

In mice, MGO induced heat hyperalgesia in a dose dependent manner when administered in

WT mice31. Moreover, STZ diabetic mice showed GLO suppression which was associated

withand hyperalgesia, and such complication was reproduced in WT mice when GLO

expression was supressed31. MGO acutely depolarises the resting membrane potential in

sensory neurons increasing their excitability. Such neuropathic events were shown to be

NaV1.8 –dependent, as these events were absent in NaV1.8 –KO mice31Molecular studies

showed MGO triggers changes in NaV1.8 gating through DIII-DIV linker’s arginine residue

modification in mice dorsal root ganglion (DRG)31.

Furthermore, MGO increases the release of calcitonin gene related peptide (CGRP) in

peripheral nerves and nerve conduction in STZ-diabetic and control mice31. These neuronal

events are also accompanied by an increase in cerebral blood flow to areas associated with

nociception31.

A further mechanism for exacerbation of diabetic neuropathy is activation by MGO of

human ankyrin transient receptor potential TRPA1. An effect which was blocked by the

TRPA1 antagonist (HC030031)13. TRPA1 is expressed in sensory neurons and mediates

nociception through numerous noxious compounds such as the highly reactive electrophile,

MGO. Binding of MGO to the cysteine and lysine residues of the channel’s N-terminal

intracellular domain channel are necessary for channel activation by MGO13.

Sensory neurons from TRPA1 knockout mouse shows no calcium influx when treated with

MGO. MGO facilitates the release of CGRP from vagus and sciatic nerves as well as from the

skin that contributes to pain transmission and together with PKA and PLC leads to nerve

sensitization. MGO might contribute to neuropathic pain in diabetes13.

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Table 1 Methylglyoxal effects on diabetes induction or diabetic complications

Model Diabetes related effect Significant findings

Sprague-Dawley rats

MGO 60mg/kg/day for 28 days induces insulin dysfunction and hyperglycaemia and therefore is concluded as diabetogenic

• Fasting plasma glucose elevation • Insulin release, GLUT-4, PI3-K and adipose

glucose uptake reduction35

Human insulin, cell culture studies on 3T3-L1 adipocytes, L8 skeletal muscle cells, H4-II-E cells and INS-1E cells.

MGO 10μM-1mM induces insulin resistance and thus is considered as diabetogenic

• Mass spectrophotometry: additional peaks of MGO-bound insulin

• L8-cells showed significant reduction in glucose uptake

• MGO binds INS-1E cells and reduces Insulin negative feedback33

RINmf5 insulin secreting cells

MGO 100μM-10mM induces cells toxicity and thus is considered as diabetogenic

• Fragmented nuclei cells elevation recorded microscopically and with multiparameter flow cytometry11

Human plasma and erythrocytes

MGO 30-300μM was shown to accelerate eryptosis

• HPLC analysis showed plasma and RBC MGO elevation

• MGO reduced GSH and ATP in RBC • MGO dose dependent annexin-V-positive

elevation46 Mouse aortic endothelial cells

MGO 500μM induces endothelial dysfunction through interfering endothelial insulin signalling

• Western blotting: Inhibiting IRS-1, Akt and eNOS phosphorylation37

Human plasma MGO derivatives were associated with endothelial dysfunction

• ELISA measurements showed sVCAM-1 and sPLA2 elevation associated with THP in T1DM patients44

STZ Wistar-Kyoto rats saphenous artery

MGO elevation was associated with vascular function which is a major complication in diabetes

• Mild impairment in cholinergic and sodium nitroprusside (SNP) induced vasodilation41

Wistar and Goto-Kakizaki rats

MGO induces endothelial dysfunction even when ingested (50-75mg/kg/day for 3 months)

• Cholinergic vasodilation significant impairment

• Aortic IHC showed significant reduction in free NO production accompanied with increase in superoxide generation

• Western blotting showed significant suppression of phosphorylated and total

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Conclusion:

vasodilator stimulated phosphoprotein • Vascular inflammation through increased

monocyte chemoattractant peptide-1 # (MCP-1)43

Normal human LDL, human BJ fibroblast AND HepG2 cells and Charles River rats

MGO-bound LDL accelerates vascular complications such as atherosclerosis

• LDL particles significantly decreased through MGO binding

• Cell free microplate blocked wells showed significant aggregation tendency of MGO-bound LDL

• IHC: MGO increased LDL retention in rats aorta

• MGO-bound LDL binds significantly more to LDL receptors found on HepG2 and BJ cells49

Sprague Dawley rat aortic rings, rat aortic and human umbilical veins endothelial cells.

MGO 100μM induces endothelial dysfunction, a common diabetes complication

• Cholinergic endothelium dependent vasodilation significant impairment accompanied with significant decrease in endothelial NO production with suppressed eNOS phosphorylation estimated through western blotting40

Mice (TRPA1+/-) HEK 293t cells and DRG cultures

MGO 10mM induces neuropathic pain, a major diabetic complication

• MGO generates large inward current in HEK 293t cells and depolarizes the membrane from -100 to +100mV

• Calcium imaging reveals MGO binding to cysteine preferably to induce calcium entry

• TRPA1 or TRPV1 KO DRG showed significant lack of response to MGO13

Diabetic patients, WT and STZ mice mice (Glo-/+), mice (Scn-/-), human sciatic nerve, mice DRG, MGO treated Wistar rats

MGO 5μM administered systemically into rats induces hyperalgesia and neuronal inflammation for 3 hours

• MGO elevation was associated with diabetic neuropathy in T2DM patients31

• MGO depolarizes sensory neurons and modifies NaV1.8 to increase the neuronal excitation and facilitate nociception

• MGO slows NaV1.7 inactivation • In mice, MGO reduces nerve conduction and

increases calcitonin gene-related peptide (CGRP) and cyclooxygenase-2 (COX-2) expression to promote thermal and mechanical hyperalgesia in mice

• MGO increases blood flow to nociceptive cerebral regions31

Transgenic GLO-1 and normal Wistar rats

Restoring MGO metabolism prevents retinopathy, a common visual complication in diabetes

• Immunoblotting showed significant increase in CEL, CML, MG-H1 in diabetic Wistar rats’ retinas suppressed in GLO1 transgenic rats45

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Diabetes mellitus is a metabolic disorder that is a major health burden in most of the

countries around the globe. Although numerous therapeutic options are available to control

diabetes, these medications are targeted mainly toward controlling the blood glucose level

through supplying insulin, enhancing insulin secretion, enhancing the tissues’ sensitivity

towards insulin, interfering with glucose absorption or re-absorption. However, diabetic

complications such as retinopathy, neuropathic pain, vascular and renal complications are

still the main diabetic complications that, in the long term, remain largely resistant to

treatments. Numerous studies reviewed in this paper show a correlation between MGO and

diabetes as well as diabetes complications which suggest that understanding the actions of

MGO might identify therapeutic targets for treating consequences of diabetes in the future.

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